Polymer Encapsulation of Bacterial Biosensors Enables Coculture with Mammalian Cells

Coexistence of different populations of cells and isolation of tasks can provide enhanced robustness and adaptability or impart new functionalities to a culture. However, generating stable cocultures involving cells with vastly different growth rates can be challenging. To address this, we developed living analytics in a multilayer polymer shell (LAMPS), an encapsulation method that facilitates the coculture of mammalian and bacterial cells. We leverage LAMPS to preprogram a separation of tasks within the coculture: growth and therapeutic protein production by the mammalian cells and l-lactate biosensing by Escherichia coli encapsulated within LAMPS. LAMPS enable the formation of a synthetic bacterial–mammalian cell interaction that enables a living biosensor to be integrated into a biomanufacturing process. Our work serves as a proof-of-concept for further applications in bioprocessing since LAMPS combine the simplicity and flexibility of a bacterial biosensor with a viable method to prevent runaway growth that would disturb mammalian cell physiology.

The starting L-lactate-biosensing plasmid used in this work (pLac) is described in Trantidou et al.,1 and is a modification of the original plasmid design of Goers et al. 2 , where the native lldPRD promoter from E. coli was exchanged for a synthetic promoter consisting of the constitutive J23117 promoter flanked by the O1 and O2 sites of the natural promoter ( Figure 1a). For the present work, the promoters J23100 and J23118 were used individually in place of the Hyperspank promoter K143015 to control the expression of the lldR transcription factor and the selection marker was changed from -lactamase (ampicillin resistance) to kanamycin phosphotransferase.
The aim of these changes was to achieve a better dose response of the system and avoid the need to add IPTG to the current experimental set up to induce lldR expression.
For the Hyperspank promoter substitution, the pLac plasmid was amplified by inverse PCR with primers P1 and P2 upstream and downstream of the promoter. The PCR product was gel extracted, purified and digested with BamHI HF and XhoI restriction enzymes (New England Biolabs, UK) and subsequently purified. The J23100 and J23118 promoter inserts were prepared by annealing primers P3 and P4 and P5 and P6, respectively. The primers were dissolved in annealing buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.5), mixed in equal volumes, warmed to 99ºC in a heat block and left to cool down to room temperature. The resulting dsDNA inserts contained unpaired ends complementary to the BamHI and XhoI restriction sites and were ligated to the previously obtained backbone using T4 ligase (Promega, UK) to obtain the plasmids pLact-S2 E. coli DH5α cells were used for expression and characterization of all the plasmids constructs. A colony of freshly transformed cells carrying the plasmid of interest was inoculated into 5 mL of LB medium supplemented with 37.5 mg/L of kanamycin and grown at 37°C with shaking at 250 rpm.
After 8-10 h, 100 µL of this starter culture was used to inoculate 10 mL of fresh M9 medium with 37.5 mg/L of kanamycin and grown for 16 h under the same conditions. Fresh M9 medium was inoculated from this culture to a starting OD of 0.05 in 200 µL cultures in 96 well flat bottom plates (Corning, USA). The plate was kept at room temperature for 2h 30 min to simulate the preparation time of the LAMPS beads. L-lactate and IPTG were added to the desired concentrations after this incubation step. The plates were covered with a Breath Easy membrane (Sigma) and incubated in a CLARIOstar plate reader (BMG Labtech, UK) at 37ºC and 500 rpm, taking fluorescence reads every 15 min with λ ex =470 nm and λ em =515 nm for the detection of GFP. The fluorescence measurements were normalized by the corresponding OD 600 measurement.

Coating with chitosan (CH) and poly-dopamine (PD)
The coating with PD followed the same protocol described in the main text, using a 5 mg/mL dopamine hydrochloride solution. The incubation was extended either to 3.5 h or 12 h.
Coating with CH followed the method by Gåserød et al. 3 A 1.5 g/L chitosan solution was prepared in a solution of 0.02M CH 3 COONa and 0.1 M CaCl 2 . The mix was acidified with HCl to pH 4, and upon dissolution of the chitosan, the pH was adjusted to 6 with NaOH and filter sterilized. The chistosan solution was used in a 1 hour incubation step for the first layer and 2 hours for the second. The rest of the steps were as described in the main text.

Analytical methodology
-Culture viability and cell density Culture viability and cell density were measured in a NucleoCounter® NC-250 (Chemometec, Germany), by staining 95 µL of cell culture with 5 µL of solution C-18 (acridine orange+DAPI, Chemometec). Prior to measurements adherent CHO cells were washed with 2 mL of PBS, incubated with 0.5 mL of prewarmed 0.05% trypsin solution at 37 ºC for 5 min on a rocking platform, followed by the addition of 1 mL of neutralizing solution (10% FBS in PBS). Suspension cells were used directly for measurements.
-L-lactate concentration L(+)-lactate concentration in the culture media was measured with an enzyme-based assay kit (MAK064, Sigma, UK) following the instruction provided by the manufacturer. For sample preparation, cells and insoluble material were removed from a 1 mL culture sample by centrifugation at 13000 g for 10 min and filtering with a 5 kDa MWCO spin filter (Vivaspin 500, Sartorius) to remove any lactate dehydrogenase contamination. Prepared samples were kept at -20 ºC until analysis. L-lactate concentration was measured spectrophotometrically using absorbance at 570 nm. Samples were diluted as needed to have a concentration between 0.05 S3 and 0.1 µmol/mL for the enzymatic reaction. A calibration curve with a standard of L-lactate was prepared to enable quantification.

-Bacterial escape from LAMPS
To test possible escape of E. coli cells from the LAMPS during incubation, 100 µL of culture medium was plated on LB-agar plates containing 37.5 mg/L kanamycin and incubated overnight at 37ºC to assess colony forming units (CFU). Plates were analysed in at least duplicate for each timepoint during the co-culture experiment and the optimization of the encapsulation protocol.  Here, the fresh M9 used for the resuspension of the cells during the preparation of the LAMPS is the only nutrient source available, therefore a higher number of layers will reduce the diffusion rate of the nutrients outside the bead. mM. Datapoints at 1, 2.6 and 5 hours were extracted from these data and are presented in Figure   3.a.